Summary This proposal focuses on UBQLN2, missense mutations in which cause dominant inheritance of amyotrophic lateral sclerosis with frontotemporal dementia (ALS-FTD). UBQLN proteins facilitate clearance of misfolded proteins from cells through the autophagy and proteasome degradation pathways, including in ER-associated degradation (ERAD). Disturbances in ERAD lead to induction of ER stress, chronic induction of which is implicated in the pathogenesis of neurodegenerative diseases, including ALS. There is growing appreciation that ER stress may be involved in ALS pathogenesis because targeting of elements that regulate this signaling response can alleviate disease. However, we not only lack good understanding of the underlying mechanisms by which mutations in ALS genes trigger ER stress, but also good animal models of the mutations. We recently generated transgenic mice lines that express either wild type (WT) or the P497S or P506T UBQLN2 mutations that cause ALS-FTD. The lines expressing the UBQLN2 mutations develop motor neuron disease and cognitive deficits, recapitulating key features of the human disease. By contrast, the WT lines are devoid of disease. Examination of protein changes in the spinal cord of early and the end-stage mutant UBQLN2 lines revealed robust elevation of ER stress and autophagy markers, suggesting protein homeostasis has been perturbed. ER stress triggers the activation of Ire1?, PERK and ATF6 signaling pathways, collectively called the unfolded protein response (UPR). There are two phases of UPR: an adaptive phase, where attempts are made to restore protein homeostasis, which, if unsuccessful, triggers a terminal cell death phase. Studies have shown that genetic or pharmacological methods that prolong the adaptive phase, or which block the cell death phase, delay disease in SOD1 mouse models of ALS. In this application we will utilize similar strategies to directly test whether modulation of UPR signaling, or autophagy, will alleviate disease in our UBQLN2 mouse models of ALS-FTD. There are six aims. In Aim 1 we will use in vitro cell and biochemical assays to gain mechanistic insight into how UBQLN2 mutations interfere with ERAD. In Aim 2 we will evaluate if prolongation of the adaptive phase of ER stress, by genetic deletion of GADD34, alleviates disease symptoms in mice carrying the P497S UBQLN2 mutation. In Aim 2 we will evaluate if genetic deletion of CHOP, a molecule involved in execution of cell death, alleviates disease in mutant P497S mice. In Aim 4 we will evaluate whether genetic deletion of ASK1, a kinase that acts downstream of Ire1? to drive cell death signaling, alleviates disease in P497S mutant mice. In Aim 5 we will assess if enhancement of autophagy will alleviate any disease in our UBQLN2 lines. In Aim 6 we will use co-culture experiments to determine if astrocytes from our UBQLN2 mouse models can induce non-cell autonomous death of MN. The results of this research will provide important insight into the underlying mechanisms by which UBQLN2 mutations cause disease, the lessons of which could be exploited for therapeutic intervention in ALS-FTD.